End-blocked peptides inhibiting binding capacity of gp120

ABSTRACT

A C- and N-blocked prolyl-alanine compound having the structure ##STR1## in which X is --OCH 2  --B or ##STR2## in which A and B are hydrogen or phenyl and Y is isobutyl inhibits binding capacity of GP120 and inhibits infection of cells by HIV-1.

This invention was made with government support and the federalgovernment has certain rights in the invention.

This invention relates to new chemical compounds and to a method ofinhibiting infection of cells with HIV as well as to a method ofinhibiting formation of syncytium from cells infected with HIV by meansof the compounds, and pertains more specifically to C- and N-blockedprolylalanine or prolylphenylalanine compounds which bind specificallyto gp 120 envelope protein of HIV-1 and inhibit binding of the envelopeprotein to CD4 as well as inhibiting formation of syncytium from cellsinfected with HIV-1.

CD4, a surface glycoprotein found primarily on a T lymphocyte subset, isa receptor both for the Class II major histocompatibility complexantigens (MHC) and for the human immunodeficiency viruses (HIV).Physiologically, CD4 is believed to bind a monomorphic domain on ClassII MHC, thereby facilitating antigen recognition and enhancing T cellactivation. The envelope glycoprotein gp 120 of HIV includes a bindingsite which binds to CD 4, and the respective binding sites of bothglycoproteins have been mapped. Binding of CD4 by gp 120 blocks theability of CD 4 to bind Class II MHC or to be stimulated by Class IIMHC, thereby interfering with effective immunological response toinfection by HIV.

It has previously been proposed to provide soluble forms of CD4 to bindto the HIV envelope protein gp 120, thus inhibiting the spread ofinfection by blocking viral adsorption and syncytium formation by thecells, while not interfering with the binding of cellular CD4 to theClass II MHC proteins. However, the use of the soluble forms of CD4 astherapeutic agents presents several problems in terms of delivery,stability, and expense.

It has now been found that certain C- and N-blocked prolylalanine andprolylphenylalanine compounds having the structure: ##STR3## in which Xis --OCH₂ --B or ##STR4## in which A and B are hydrogen or phenyl, and Yis isobutyl

are capable of binding to the envelope or surface glycoprotein gp 120 ofHIV. This binding inhibits the binding of the HIV to the CD4 surfaceglycoprotein of T cells, thus inhibiting infection of the cells by thevirus; in addition the new compounds bind specifically to the gp 120glycoprotein present on the surface of previously infected cells,inhibiting syncytium formation by those cells and in consequence furtherinfection by the already infected cells. It also reverses the effect ofgp 120 in inhibiting the binding of CD4 to Class II MHC.

The binding of the inhibitor compounds to the HIV or to cells infectedwith the HIV is accomplished simply by bringing a solution or dispersioncontaining one or more of the compounds into contact with HIV or with acell infected with HIV. Any appropriate medium or carrier, preferablyaqueous, can be used in the solution or dispersion.

The stereochemistry of the peptide backbone of the compounds has littleeffect upon the binding activity of the compound; all four possiblestereoisomers of the compounds have been found to have very similarbinding activities. However, the enantiomer which contains the D-isomersof both proline and phenylalanine exhibits the greatest activity and ispreferred. However, replacement of the benzyl moiety with a methylmoiety in the C-blocking group results in substantial loss of bindingactivity as does replacement of the methoxydicarbonyl blocking group bytertiary butoxycarbonyl (BOC). Substitution of an alanyl moiety for thephenylalanyl moiety also results in substantial reduction of bindingactivity.

EXAMPLE 1

The compounds of the invention were made by conventional chemicalprocedures utilizing the appropriate amino acids, proline, alanine,phenylalanine and leucine in D- or L- form, and using conventionalblocking agents.

In general, N-blocked BOC-proline was prepared from D- or L- proline,and N-blocked BOC-alanine or BOC-phenylalanine was also prepared from D-or L- alanine after which the BOC-alanine or BOC-phenylalanine wasesterified with benzyl alcohol or methyl alcohol, as the case may be.The BOC blocking group was then removed from the esterified alanine orphenyalanine with trifluoracetic acid (TFA) and the product was coupledwith the BOC-proline. Finally, the BOC blocking group on the proline wasremoved with TFA and the product was reacted with methyloxalyl chloride.In the case of the compound in which X is ##STR5## where Y is isobutyland B is phenyl, the analogous procedure was followed by esterifyingBOC-protected leucine with benzyl alcohol, then coupling with the otheramino acids. The products were purified by recrystallization. Therecrystallized inhibitors were dissolved in DMSO at 8 mg/ml. On the dayof the assay the inhibitors were diluted 10-fold into PBS and 2-foldserial dilutions made into PBS previously adjusted to the same DMSOconcentration. 25 μl each of inhibitor solution and 20 μg/ml gp 120(from baculovirus; MicroGeneSys) were combined and incubated at 37° C.for 1 hour. 3-5×10⁵ HSBCD4-M.23 cells (from a clone of the pre-T cellleukemia line HSB-2 which expresses CD4) were pelleted in the wells of a96-well V-bottom microtiter plate, resuspended with the preincubated gp120-inhibitor mixture and allowed to incubate at 4° C. for 1 hour. Thecells were then pelleted and resuspended in 50 μl of rabbit anti-gp120(1:400 in PBS; MicroGeneSys) and incubated at 4° C. for 30 min. Thecells were then pelleted, washed with 100 μl PBS and resuspended influorescein isothiocyanate conjugated goat anti-rabbit Ig (1:20:Tago)and incubated at 4° C. for 30 min. The cells were pelleted and washed asbefore, and resuspended in 100 μl of 25 μg/ml propidium iodide andtransferred to tubes containing 400 μl PBS for flow cytometric analysison an Epics V (Coulter) for RACScan (Becton-Dickenson). Cells were gatedon propidium iodide to exclude dead cells. Green fluorescence wascollected on the linear amplifier and the mean fluorescence calculated.Values were corrected by subtracting the mean fluorescence of cellstreated with both antibodies but not exposed to gp120, which did notdiffer significantly from the intrinsic fluorescence of the cells, andwas generally <15. 10 μg/ml is a subsaturating concentration for thebaculovirus-produced gp120 used and the relation between fluorescenceintensity and gp120 concentration approaches linearity.

The following inhibitor compounds were tested, referring to the generalformula set forth above:

1. X is --OCH₂ --B, A and B are phenyl, both amino acids are in theL-form;

2. X is --OCH₂ --B, A is phenyl, B is hydrogen;

3. X is ##STR6## A and B are phenyl, and Y is isobutyl; 4. X is --OCH₂--B, A is hydrogen and B is phenyl;

5. X is --OCH₂ --B, A and B are phenyl, both amino acids are in theD-form.

In each case the corrected mean fluorescence decreased as theconcentration of the inhibitor increased from about 50 μM to 900 μM,showing increasing inhibition of binding to CD4 by the gp 120 which hadbeen preincubated with an inhibitor compound.

EXAMPLE 2

100 μl solutions containing 20 μg/ml gp120 with and without 200 μg/ml ofcompound 5 above were incubated at 37° C. for 1 hour. 50 μl of each wasdiluted to 1 ml with PBS and then reconcentrated by ultrafiltration (20min., 5.7 krpm in an SA-600 rotor; Dupont) in a Centricon-30 (molecularweight cutoff=30,000; Amicon). The Centricons were pretreated with 1%casein to reduce non-specific binding of gp120 to the device. Theundiluted 50 μl aliquots, as well as a sample containing gp 120+10 μg/ml(the concentration of free inhibitor after ultrafiltration), were storedat 4° C. during ultrafiltration. These samples and the recoveredretentates were then used to resuspend HSBCD4-M.23 and assayed asdescribed in Example 1 above. Binding to the CD4 by the gp 120pretreated as described above is inhibited to a similar extent bothbefore and after dilution and ultrafiltration, as compared to untreatedgp 120. Moreover, the extent of inhibition is greater than was obtainedin Example 1 at the same concentration as the residual, showing that theinhibitor binds to and remains bound to the gp 120, and showing that thebinding is not rapidly reversible, perhaps because the inhibitors arepoorly soluble in water.

EXAMPLE 3

The physiologic ligands of CD4 are the Class II MHC proteins. Thisinteraction is well known to promote conjugate formation in vitrobetween human CD4⁺ murine T cell hybridomas and Class II MHC⁺ cells.Conjugate formation with Class II MHC⁺ cells can also be observed withthe HSBCD4-M.23 cells of Example 1. It has also been previously knownthat gp 120 inhibits conjugate formation. This inhibition is reversed byboth compound 1 and compound 5, but not by compound 4 or compound 3.Inhibition of conjugates is observed either with preincubation of gp 120and compound 1 or 5 or with simultaneous addition of components. Thesecompounds have no effect on conjugate formation directly. Thus compounds1 and 5 would not be expected to interfere with this aspect of T cellfunction. To the contrary they would reverse any inhibition due to gp120.

Conjugate formational assays were performed using the CD4⁺ HSBCD4-M.23and Class II MHC⁺ cell lines (Daudi). To evaluate the cell-cellconjugate formation the HSBCD4-M.23 cells were labeled with 0.5 μg/mlsulfofluorescein diacetate (Molecular Probes, Eugene, Oreg.) and Daudicells were labeled with 40 ng/ml hydroethididne (Polysciences, Inc.,Warrington, Pa.) by conventional methods. Cells at 1×10⁶ /ml were mixedat a 1:2 CD⁴⁺ /Class II MHC⁺ cell ratio and incubated at 37° C. for ≧60min. in 30 μl or RPMI 1640, 10% fetal calf serum in 1.5 mlmicrocentrifuge tubes. Inhibitors, gp 120, and their mixtures were addedsimultaneously with the cells. Preincubation of gp 120-inhibitormixtures was at 37° C. for 20-30 min. prior to addition. At the end ofthe incubation period samples were gently resuspended by repipetting 5times through a tip having an internal diameter of 0.5 mm. To quantitatethe percent of conjugates formed a 9 μl aliquot was placed on a glassslide and a 12 mm coverslip was placed over the sample and held in placewith fingernail polish. Observations were made with a light microscopeusing a fluorescent lamp to locate the CD4⁴⁺ fluorescein cells. Aconjugate was defined as at least two cells of one color bound to atleast one of the other. Blinded samples were run in duplicate and threeor more counts of at least 100 fluorescein-labeled cells were done foreach sample and scored as conjugates or non-conjugates. The percent ofconjugates was calculated as the total number of conjugates divided bythe total number of free CD4⁺ cells plus conjugates. Results areexpressed as the mean value ±SEM of the six counts made for each group.

EXAMPLE 4

HIV-1 (strains HILViii_(B) or MN) was derived from filtered (0.54μ)supernatants of H9 infected cells. Supernatants were incubated withcompound 5 at 100 μm for 1 hour at 37° C. One ml specimens of the virussupernatant (with or without compound 5 at 100 μM) were incubated with10⁶ H9 cells at a multiplicity of infection of 10. Cells were washedthree times and carried at 10⁵ cells/ml in RPMI 1640 and 10% Fetal CalfSerum. Supernatants were analyzed at three day intervals up to a totalof 15 days for production of p24 antigen by ELISA assay. No p24 antigenwas found in the specimens containing compound 5, showing that compound5 inhibits HIV infection of CD4+ T cells.

Similar results can be obtained with compounds in which one amino acidis in D-, the other in L-form.

The compounds, in a suitable pharmacologically acceptable non-toxiccarrier can be administered to mammals such as man orally or byintravenous or intraarterial injection to inhibit infection by HIV.

What is claimed is:
 1. A C- and N-blocked prolyl-alanine compound havingthe structure ##STR7## in which X is --OCH₂ --B or ##STR8## in which Aand B are hydrogen or phenyl and Y is isobutyl.
 2. A compound as claimedin claim 1 in which both the prolyl and the alanyl moieties are in theD-form.
 3. A compound as claimed in claim 1 in which X is --OCH₂ --B andA and B are phenyl.
 4. A compound as claimed in claim 3 in which boththe prolyl and the alanyl moieties are in the D-form.